Pranchevicius M.-C.S.,FFCLRP |
Oliveira L.L.,FMRP |
Oliveira L.L.,Federal University of Viçosa |
Rosa J.C.,FMRP |
And 4 more authors.
BMC Biotechnology | Year: 2012
Background: ArtinM is a d-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system.Results: The ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized d-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises β-sheet structure.Conclusions: Overall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin. © 2012 Pranchevicius et al.; licensee BioMed Central Ltd.
Costa F.H.S.,FFCLRP |
Campos M.,São Paulo State University |
da Silva M.A.A.,FFCLRP
Journal of Theoretical Biology | Year: 2015
In this work, we used five cell lineages, cultivated in vitro, to show they follow a common functional form to the growth rate: a sigmoidal curve, suggesting that competition and cooperation (usual mechanisms for systems with this behavior) might be present. Both theoretical and experimental investigations, on the causes of this behavior, are challenging for the research field; since the sigmoidal form to the growth rate seems to absorb important properties of such systems, e.g., cell deformation and statistical interactions. We shed some light on this subject by showing how cell spreading affects the radius behavior of the growing colonies. Doing numerical time derivatives of the experimental data, we obtained the growth rates. Using reduced variables for the time and rates, we obtained the collapse of all colonies growth rates onto one curve with sigmoidal shape. This suggests a universal-type behavior, with regime transition related to a morphological transition of adherent cell colonies. © 2015 Elsevier Ltd.